scholarly journals Stopped-flow fluorescence kinetics of bovine α2-antiplasmin inhibition of bovine midiplasmin

1995 ◽  
Vol 305 (1) ◽  
pp. 97-102 ◽  
Author(s):  
S Christensen ◽  
L Sottrup-Jensen ◽  
U Christensen
Keyword(s):  
Binding Site ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Reaction Model ◽  
Stopped Flow ◽  
Reaction Step ◽  
Fast Kinetics ◽  
Autocatalytic Cleavage ◽  
Series Of Experiments ◽  
The One

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.

scholarly journals Probing an Open CFTR Pore with Organic Anion Blockers

2002 ◽  
Vol 120 (5) ◽  
pp. 647-662 ◽  
Author(s):  
Zhen Zhou ◽  
Shenghui Hu ◽  
Tzyh-Chang Hwang
Keyword(s):  
Binding Site ◽  
Voltage Dependence ◽  
Organic Anion ◽  
Kinetic Studies ◽  
Transmembrane Conductance Regulator ◽  
Fast Kinetics ◽  
Slow Kinetics ◽  
Kd Values ◽  
Voltage Dependent ◽  
Physical And Chemical

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl− current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose–response relationships of both blockers follow a simple Michaelis-Menten function with Kd values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl− increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl− binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl− concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl− and isethionate compete for a binding site in the pore. The voltage dependence and external Cl− concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.

Global Reaction Model to Describe the Kinetics of Catalytic Pyrolysis of Coffee Grounds Waste

2017 ◽  
Vol 899 ◽  
pp. 173-178 ◽  
Author(s):  
Ronydes Batista Jr. ◽  
Bruna Sene Alves Araújo ◽  
Pedro Ivo Brandão e Melo Franco ◽  
Beatriz Cristina Silvério ◽  
Sandra Cristina Danta ◽  
...  
Keyword(s):  
Thermal Decomposition ◽  
Large Scale ◽  
Catalytic Pyrolysis ◽  
Petroleum Products ◽  
Reaction Model ◽  
Heating Rates ◽  
Coffee Grounds ◽  
One Step ◽  
Global Reaction ◽  
The One

In view of the constant search for new sources of renewable energy, the particulate agro-industrial waste reuse emerges as an advantageous alternative. However, despite the advantages of using the biomass as an energy source, there is still strong resistance as the large-scale replacement of petroleum products due to the lack of scientifically proven efficient conversion technologies. In this context, the pyrolysis is presented as one of the most widely used thermal decomposition processes. The knowledge of aspects of chemical kinetics, thermodynamics these will, heat and mass transfer, are so important, since influence the quality of the product. This paper presents a kinetic study of slow pyrolysis of coffee grounds waste from dynamic thermogravimetric experiments (TG), using different powder catalysts. The primary thermal decomposition was described by the one-step reaction model, which considers a single global reaction. The kinetic parameters were estimated using nonlinear regression and the differential evolution method. The coffee ground waste was dried at 105°C for 24 hours. The sample in nature was analyzed at different heating rates, being 10, 15, 20, 30 and 50 K/min. In the catalytic pyrolysis, about 5% (w/w) of catalyst were added to the sample, at a heating rate of 30 K/min. The results show that the one-step model does not accurately represent the data of weight loss (TG) and its derivative (DTG), but can do an estimative of the activation energy reaction, and can show the differences caused by the catalysts. Although no one can say anything about the products formed with the addition of the catalyst, it would be necessary to micro-pyrolysis analysis, we can say the influence of the catalyst in the samples, based on the data obtained in thermogravimetric tests.

scholarly journals Kinetic Studies of Proton Transfer in the Microenvironment of a Binding Site

1982 ◽  
Vol 121 (3) ◽  
pp. 637-642 ◽  
Author(s):  
Menachem GUTMAN ◽  
Dan HUPPERT ◽  
Esther NACHLIEL
Keyword(s):  
Proton Transfer ◽  
Binding Site ◽  
Kinetic Studies

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

I. On the volumetric relations of ozone and the action of the electrical discharge on oxygen and other gases

1860 ◽  
Vol 10 ◽  
pp. 427-428 ◽  
Keyword(s):  
Electrical Discharge ◽  
The Other ◽  
Pure Oxygen ◽  
Silent Discharge ◽  
Gaseous Substance ◽  
Series Of Experiments ◽  
The One

This paper contains the full details of the authors’ experiments on the volumetric changes which occur in the formation of ozone. From three distinct series of experiments, performed by different methods, they show that when ozone is formed from pure oxygen by the action of the electrical discharge, a condensation takes place, as had already been announced in a former Note published in the 'Proceedings.’ But the condensation is much greater than the earlier experiments of the authors on the expansion by heat of electrolytic ozone had indicated. It is, in fact, so great, that if the allotropic view of the constitution of ozone be correct, the density of that body, as compared with oxygen, would be represented by a number corresponding to the density of a solid or liquid rather than that of a gaseous substance. This conclusion follows necessarily from the authors’ experiments, unless it be assumed that when ozone comes into contact with such substances as iodine, or a solution of iodide of potassium, one portion of it is changed back into common oxygen, while the remainder enters into combination, and that these portions are so related to one another, that the expansion due to the one is exactly equal to the contraction arising from the other. For the details of the experiments and of the methods of investigation employed, reference must be made to the original paper. The second part of the communication is devoted to the action of the silent discharge and of the electrical spark on other gases. Hydrogen and nitrogen undergo no change of volume when exposed to the action of either form of discharge. Cyanogen is readily decomposed by the spark, but presents so great a resistance to the passage of electricity, that the action of the silent discharge can scarcely be observed. Protoxide of nitrogen is readily attacked by both forms of discharge, with increase of volume and formation of nitrogen and hyponitric acid. Deutoxide of nitrogen exhibits the remarkable example of a gas which, under the action either of the silent discharge or of the spark, undergoes, like oxygen, a diminution of volume. It also is resolved into nitrogen and hyponitric acid. Carbonic oxide has given results of great interest; but the nature of the reaction has been only partially investigated. The silent discharge decomposes this gas with production of a substance of a bronze colour on the positive wire. The spark acts differently, destroying, as in the case of oxygen, the greater part of the contraction produced by the silent discharge. The authors are engaged in the further prosecution of this inquiry.

scholarly journals Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families

2017 ◽  
Vol 114 (7) ◽  
pp. E1091-E1100 ◽  
Author(s):  
Mario D. Garcia ◽  
Amanda Nouwens ◽  
Thierry G. Lonhienne ◽  
Luke W. Guddat
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Kinetic Studies ◽  
Herbicidal Activity ◽  
Structural Basis ◽  
Acetohydroxyacid Synthase ◽  
Branched Chain Amino Acid ◽  
Application Rates ◽  
Herbicide Binding ◽  
Branched Chain

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted “S”-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.

Observation of a radical intermediate in the one electron oxidation of 5-methyl-1-thia-5-azacyclooctane by •Br2−

1984 ◽  
Vol 62 (9) ◽  
pp. 1874-1876 ◽  
Author(s):  
Warren Kenneth Musker ◽  
Parminder S. Surdhar ◽  
Rizwan Ahmad ◽  
David A. Armstrong
Keyword(s):  
Aqueous Solution ◽  
Rate Constant ◽  
Half Life ◽  
Cation Radical ◽  
Order Rate Constant ◽  
Type Structure ◽  
Radical Intermediate ◽  
Text Type ◽  
First Order ◽  
The One

The one electron oxidant •Br2− reacts with 5-methyl-1-thia-5-azacyclooctane (4) in aqueous solution at high pH with an overall rate constant of ~2 × 108 M s−1. The radical intermediate produced has a broad maximum at 500 nm with ε = 2400 M−1 cm−1 and at pH 10 decays with a first order rate constant of 2.3 ± 0.3 × 104 s−1, first half-life of 30 ± 5 μs. Its characteristics do not correspond to those of the [Formula: see text] species reported by Asmus and co-workers. The species appears to be the same as the cation radical reported earlier in the one electron oxidation of 4 in acetonitrile. This species is considered to have an [Formula: see text] type structure, which provides transannular stabilization.

scholarly journals Qualitative analysis of an autocatalytic chemical reaction model with decay

2014 ◽  
Vol 144 (2) ◽  
pp. 427-446 ◽  
Author(s):  
Jun Zhou ◽  
Junping Shi
Keyword(s):  
Steady State ◽  
Chemical Reaction ◽  
Reaction Diffusion ◽  
Steady State Solution ◽  
Reaction Model ◽  
Existence Of A Solution ◽  
Positive Steady State ◽  
Chemical Reaction Model ◽  
The One ◽  
Necessary And Sufficient

In this paper, we revisit a reaction—diffusion autocatalytic chemical reaction model with decay. For higher-order reactions, we prove that the system possesses at least two positive steady-state solutions; hence, it has bistable dynamics similar to the system without decay. For the linear reaction, we determine the necessary and sufficient condition to ensure the existence of a solution. Moreover, in the one-dimensional case, we prove that the positive steady-state solution is unique. Our results demonstrate the drastic difference in dynamics caused by the order of chemical reactions.

scholarly journals Structure of theMycobacterium tuberculosisd-Alanine:d-Alanine Ligase, a Target of the Antituberculosis Drug d-Cycloserine

2010 ◽  
Vol 55 (1) ◽  
pp. 291-301 ◽  
Author(s):  
John B. Bruning ◽  
Ana C. Murillo ◽  
Ofelia Chacon ◽  
Raúl G. Barletta ◽  
James C. Sacchettini
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Kinetic Studies ◽  
Structural Rearrangement ◽  
Peptidoglycan Biosynthesis ◽  
Binding Cavity ◽  
Binding Pockets ◽  
Loop Regions ◽  
Discrete Domains ◽  
Alanine Dipeptide

ABSTRACTd-Alanine:d-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of twod-alanine (d-Ala) molecules to form thed-alanyl:d-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development.d-Cycloserine (DCS), an analog ofd-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure ofMycobacterium tuberculosisDdl at a resolution of 2.1 Å. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. TheM. tuberculosisapo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide andd-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP andd-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed ford-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC50) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.

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